Cells maintain a dynamic equilibrium of tension with their microenvironment and this `mechanoreciprocity' controls a wide variety of cellular functions, including cell fate, shape, and movement. Despite this importance, the molecular mechanisms through which cells sense and respond to the mechanical nature of the extracellular matrix are not completely understood. Our laboratory established that the cAMP-dependent protein kinase (PKA) is enriched and activated in the leading edge of cells and that this localization is important for cell migration. In our ongoing efforts to elucidate the mechanis for the spatial regulation of PKA during cell migration, we have recently found that localized PKA activity is regulated by cellular tension. Specifically, leading edge PKA activity is rapidly lost upon inhibition of actomyosin contractility. Moreover, when cells are mechanically stretched, PKA is rapidly and locally activated - in a tension-dependent manner - in the direction of stretch. Finally, inhibition of PKA also blocks durotaxis - cell migration guided by gradients in ECM rigidity and cell-matrix tension. Our current focus is to understand the mechanism that couples cellular tension to localized regulation of PKA. Recent preliminary data establishes that both cellular contractility and localized activation of PKA are dependent on influx of extracellula Ca2+ via the stretch-activated channel TRPM7. Additional data strongly suggest that a G- protein coupled receptor - the A2B adenosine receptor (ADORA2B) - also plays an important role in this mechanism. Based on our observations, we hypothesize that localized activation of PKA in the leading edge of migrating cells is regulated by a mechano-chemical mechanism involving interplay between localized increases in cellular tension, influx of extracellular Ca2+, and activation of ADORA2B. We will test this hypothesis by determining the role of ADORA2B in mechanical activation of PKA during cell migration, delineating the mechanism of mechano-chemical activation/regulation of ADORA2B during cell migration, and determining the mechanistic hierarchy of contractility, Ca2+, and ADORA2B in regulating localized PKA activity and cell migration. At the end of the proposed studies, we will have established a paradigmatic pathway in which localized coupling of cellular mechanics to a GPCR signaling cascade regulates cytoskeletal dynamics and cell motility.